The present invention relates to immobile buffer polymers, co-immobile buffer and enzyme polymers (or polymers in which an enzyme and a buffer are immobilized) and to methods of synthesis of polymers including immobilized buffer and polymers including immobilized enzyme and immobilized buffer.
In general, the function of an enzyme is to catalyze chemical reactions. Enzymes have a wide range of applications. For example, industrial applications of enzymes include, but are not limited to, fermenting wine, leavening bread, curdling cheese, and brewing beer. Medical applications of enzymes include, but are not limited to, killing disease-causing microorganisms, promoting wound healing, and diagnosing certain diseases.
In general, six classes of enzymes are recognized, which include oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases. Oxidoreductases catalyze oxidation or reduction reactions and are referred to as Enzyme Class 1 enzymes (EC1). Transferases catalyze the transfer of specific radicals or groups and are referred to EC 2 enzymes. Hydrolases catalyze hydrolysis reactions and are referred to as EC 3 enzymes. Lyases catalyze removal from or addition to substrate specific chemical groups and are referred to as EC 4 enzymes. Isomerases catalyze isomeration reactions and are referred to as EC 5 enzymes. Ligases catalyze reactions which combine or bind together substrate units and are referred to as EC 6 enzymes. These classifications cover generally all enzymes used in industrial, medical, and other applications.
The biocatalytic activity of enzymes in industrial, medical or other applications occurs within a range of environmental conditions (for example, pH, temperature, pressure, and ionic strength) similar to the typical biological environment of the enzymes. Each enzyme within the six enzyme classifications has, for example, an optimal pH range, in which the rate of enzyme catalyzed reaction is the fastest. This optimal pH range (as well as optimal ranges for other environmental conditions) can be narrow or broad, depending on the enzyme. Typically, plant-based enzymes have broader pH ranges than animal-based enzymes.
One method of maintaining optimal pH is to neutralize acid or base in solution. Buffer salts, for example, can be added to xe2x80x9cinitializexe2x80x9d the pH or add buffer capacity to solution. In fact, addition of buffer is the most common method for maintaining high catalytic rates for laboratory and industrial enzymatic reactions. Hydrolase enzymes (EC 3) are of special interest because they produce acid or base as a byproduct and, thus, can alter the pH of an unbuffered solution during the course of a reaction. Examples of such hydrolases includes pectinase, protease, urease, and organophosphorous hydrolase (OPH). Pectinases break down cell walls to clarify fruit juices. Proteases in detergents break down protein-based stains. Urease breaks down urea in urine into carbon dixoide and ammonia. OPH degrades organophosphates into byproducts. Pectinases, proteases, and OPH create an acid (H3O+) as byproduct. Urease creates a lewis base (NH4+) as byproduct.
Over time, the tertiary or three-dimensional structure of an enzyme may erode and the enzyme (and its active site) may correspondingly lose integrity, especially at high temperatures. This process is called enzyme denaturation. The enzyme and its active site are in proper conformation when ideal physiological conditions of, for example, moderate temperature, pH, and ionic strength are present. Seemingly minor changes in these conditions may cause changes in protein folding, resulting in catalytic activity loss and permanent denaturation. To protect against denaturation, enzymes can be immobilized on or within a solid polymer to rigidify and stabilize enzyme chemical structure. Through chemical modification, immobilization masks sensitive residues in the enzyme""s protein structure. Immobilized enzymes maintain their activity over a broader set of environmental conditions than native enzymes, including residence time in solution and temperature. By attaching an enzyme within a polymeric matrix as described, for example, in LeJeune, K. E., Mesiano, A. J., Bower, S. B., Grimsley, J. K., Wild, J. R., Russell, A. J., Biotechnol. Bioeng. 54, 105 (1997) (sometimes referred to herein as xe2x80x9cLeJeune et al. (1997)xe2x80x9d), enzyme stability is greatly enhanced.
Immobilization does not, however, protect enzymes against activity loss associated with extreme pH environments (that is, environments in which the pH is outside of the optimal pH range or well outside the optimal pH range). Both native and immobilized enzymes lose activity when placed in extreme pH environments or when significant quantities of acidic or basic byproducts are produced. For example, immobilized urease will continue to degrade urea into ammonium ion byproduct in distilled water, but when sufficient quantities of byproduct are generated, the pH rises. Immobilized urease loses activity as pH levels rise above the optimal pH and loses all activity at around pH 10. A similar effect is observed with OPH, which will degrade methyl parathion and generate acid byproduct in an unbuffered environment until reaching pH 4.
In general, immobilized enzymes are of limited use in catalyzing reactions in environments in which pH is outside of the optimal pH range of the enzyme. Moreover, immobilized hydrolases (EC 3) are of limited use in catalyzing reactions in unbuffered environments. Thus, immobilized enzymes require an added buffer to neutralize acid or base when placed in a solution with a pH outside the active range or the optimal pH range of the immobilized enzyme. Immobilized hydrolases require an added buffer to neutralize acid or base byproducts, which alter the pH to a pH outside of the active range or the optimal range of the immobilized hydrolase enzyme. However, it may not be practical or possible to add buffer to an environment in which an immobilized enzyme is to be used. For example, in field use for decontamination of a toxic agent or agents in which a large area must be decontaminated, addition of buffer may not be practical and/or buffer may not be available. Currently available immobilized enzymes cannot, therefore, be used to their full advantage in certain environments.
It is desirable, therefore, to develop immobilized enzyme systems and methods that reduce or, preferably, eliminate the above and other problems.
In one aspect, the present invention provides a polyurethane polymer including at least one buffer selected to adjust pH to a pH within a desired range. The buffer compound is immobilized within the polymer, and the polymer has a buffer capacity in excess of 3 micromoles of acid or base per gram polymer. Preferably, the polymer has a buffer capacity in excess of 60 micromoles acid or base per gram polymer. More preferably, the polyurethane polymer has a buffer capacity in excess of 100 micromoles acid or base per gram polymer. Even more preferably, the polyurethane polymer has a buffer capacity in excess of 200 micromoles acid or base per gram polymer. As described further below, attempts to incorporate significant buffer capacity in polyurethane polymers have been unsuccessful. The immobile buffer polyurethanes of the present invention provide polymers having significant buffer capacity over a wide range of polymer physical characteristics (for example, density and pore size).
In another aspect, the present invention provides a method for preparing a polyurethane polymer immobilizing at least one buffer comprising the steps: reacting a buffer compound with a multifunctional precursor for the polyurethane polymer to produce a modified precursor, the buffer compound having at least one functional group for reacting with the precursor and at least one buffering group that remains functional as a buffer after the buffer compound is reacted with the precursor; and subsequent to reacting the buffer compound with the precursor, polymerizing the modified precursor to form the polyurethane polymer.
The polyurethane polymer can, for example, be formed by first reacting the buffer compound with an isocyanate functionalized polyurethane precursor to produce a modified polyurethane precursor. Water and the modified polyurethane precursor can be mixed to form a polyurethane foam. In general, buffer immobilizing polyurethanes of a wide range of polymer physical properties can be synthesized in the present invention using modified polyurethane precursors as described above and known polymerization techniques.
In a further aspect, the present invention provides a polymer including at least one enzyme that is selected to catalyze a reaction of a substance. The enzyme is immobilized within the polymer. The polymer also includes at least one buffer selected to adjust the pH in the vicinity of the enzyme to a pH within a desired range. The buffer compound is also immobilized within the polymer. The polymer preferably has greater than 2% enzyme activity retention. In that regard, the polymers of the present invention generally incorporate approximately 0.01 to 5 wt % enzyme. Of the total enzyme loaded, preferably at least 2% of enzyme activity is retained as compared to the activity of the native enzyme. The polymer also preferably has a buffer capacity in excess of 3 micromoles acid or base per gram polymer. In one embodiment, each of the enzyme(s) and the buffer(s) are covalently bonded to the polymer. The enzyme and buffer content of such co-immobile buffer and enzyme polymer of the present invention can be tailored for use in specific environments. In one embodiment, the polymer can, for example, preferably have greater than 8% enzyme activity retention, and a buffer capacity in excess of 60 micromoles acid or base per gram polymer. In another embodiment, the polymer preferably can have greater than 15% enzyme activity retention and a buffer capacity in excess of 3 micromoles acid or base per gram polymer. In still another embodiment, the polymer can preferably have greater than 2% enzyme activity retention and a buffer capacity in excess of 200 micromoles acid or base per gram polymer.
In several embodiments, the polymer is a polyurethane. The polyurethane can, for example, be a foam having an average pore size of at least approximately 0.1 mm. More preferably, the polyurethane foam has an average pore size of at least approximately 0.2 mm. In general, average pore size is preferably sufficiently large to enable diffusion of substrate into pores to interact with immobilized enzyme and sufficiently large to enable diffusion of reaction products out of pores. The polyurethane foam preferably has a density no greater than approximately 0.4 g/cm3 . More preferably, the polyurethane foam has a density no greater than approximately 0.2 g/cm3.
In another aspect, the present invention provides a system for catalyzing a reaction of at least one substance in an environment The system includes at least one enzyme that is selected to catalyze a reaction of the substance and at least one buffer compound selected to adjust the pH in the vicinity of the enzyme to a pH within a desired range. Each of the enzyme and the buffer compound are covalently bonded within a single polymer. The polymer preferably has greater than 2% enzyme activity retention, and a buffer capacity in excess of 3 micromoles acid or base per gram polymer. In one embodiment, the single polymer is a polyurethane as described above.
In a further aspect, the present invention provides a method for preparing a polymer immobilizing at least one enzyme and at least one buffer including the steps: reacting a buffer compound with a multifunctional precursor for the polymer to produce a modified precursor, the buffer compound having at least one functional group for reacting with the precursor and at least one buffering group that remains functional as a buffer after the buffer compound is reacted with the precursor; and subsequent to reacting the buffer compound with the precursor, polymerizing the modified precursor in the presence of the enzyme to bond the enzyme to the polymer.
Once again, the polymer can be a polyurethane. In one such embodiment, the polymer is formed by first reacting the buffer compound with an isocyanate functionalized polyurethane precursor to produce a modified polyurethane precursor. The enzyme, water and the modified polyurethane precursor can, for example, be mixed to form a foamed polyurethane polymer.
In still another aspect, the present invention provides a method for preparing a modified polymer precursor for synthesis of a buffer immobilizing polymer including the step of reacting a multifunctional buffer compound with a multifunctional precursor for the polymer to covalently bond the buffer compound to the precursor compound via reaction of one of the functional groups of the buffer compound with one of the function groups of the polymer precursor, thereby producing the modified polymer precursor. The attached buffer compound has at least one functional group remaining after attachment to or incorporation within the modified polymer precursor that retains buffer capacity. The modified polymer precursor retains at least one functional group thereon suitable to react in a subsequent polymerization to synthesize the buffer immobilizing polymer. The multifunctional polymer precursor can, for example, include at least one isocyanate group. The multifunctional polymer precursor can also, for example, include at least two isocyanate groups.
As used herein, the phrase xe2x80x9cimmobile buffer polymer refers to a polymer in which on or more buffers are immobilized. As used herein, the phrase xe2x80x9cco-immobile buffer and enzymexe2x80x9d and/or the phrase xe2x80x9cco-immobile buffer and enzyme polymerxe2x80x9d refer to a polymer in which both one or more buffers and one or more enzymes are immobilized. The co-immobile buffer and enzyme polymers of the present invention are capable of buffering acid or base from solution, thereby returning the solution to a pH within a desired range. Moreover, the, co-immobile buffer and enzyme polymers of the present invention are preferably reusable, not losing significant buffer capacity or significant enzyme activity following repeated consecutive uses. In the presence of one or more immobilized enzymes, a co-immobilized buffer or buffers also serve to neutralize acidic or basic products (for example, from hydrolysis reactions) that would otherwise negatively impact enzyme performance.
By effectively incorporating immobilized buffer and enzyme together in the polymers of the present invention, the resultant buffered enzymes have high buffer capacity and high activity in the environments having a range of initial pH.
To be immobilized within a polymer, buffers preferably have at least one reactive functional group suitable to form a connecting interaction within the product macromer or polymer and at least one remaining (that is, remaining after such a connecting interaction is formed) buffering functional group suitable to neutralize acid or base in solution. Preferably, the buffer includes a functional group that is suitable to form a covalent bond within the polymer. However, the buffer can also be immobilized within the polymer via non-covalent linkages such as via strong van der waals interactions or via an ionic interaction or bond. As known in the art, to be immobilized as described herein, enzymes preferably retain significant activity following immobilization.
Regardless of the manner of immobilization, the present inventors have discovered that the overall immobilized buffer capacity of a polymer incorporating immobilized buffer can be substantially increased as compared to prior attempts to immobilize buffer or co-immobilize buffer and enzyme within a polymer matrix by first interacting or reacting the buffer with an immobilizer/stabilizer suitable to form an interactive connection (for example, a covalent bond) with buffer prior to polymer or macromer formation. Preferably, the immobilizer/stabilizer is a precursor for the product polymer. The resultant modified polymer precursor is subsequently reacted in a known manner (or polymerized) to form the desired polymer product.
It is believed that the reactivity of the buffer alters the chemical reactions occurring during polymer or macromer formation when one attempts to incorporate buffer into a polymer during polymer formation as done in current synthetic procedures. In other words, the high degree of reactivity between buffer and polymer precursor(s) (that is, a monomer, a dimer, an oligomer (generally, a molecule having less than ten repeat units), or a prepolymer (generally, a molecule having a number average molecular weight of less than 50,000)) is believed to alter the polymerization process in current synthetic procedures. As a result, the polymers synthesized in such current synthetic procedures have poor physical characteristics, low buffer capacity and poor enzyme activity in environments of uncontrolled or unbuffered pH.
To the contrary, the immobile buffer polymers and the co-immobile buffer and enzyme polymers of the present invention, exhibit both desirable physical characteristics and retain high buffer capacity. In general, the incorporation of buffer or the incorporation of buffer and enzyme into the polymers of the present invention does not substantially alter the underlying polymerization as compared to standard polymerizations (that is, polymerizations without immobilize buffer or enzyme). The stabilization of the buffer via, for example, forming an interactive connection or bond with the polymer precursor reduces the reactivity of the buffer during the polymerization step and allows the reaction to proceed in a manner in which desirable polymer properties can be achieved in a controlled manner.
Moreover, co-immobile buffer and enzyme polymers of the present invention can be prepared in a wide variety of physical forms and morphologies such as fabrics, foams, gels, rubbers, and plastics using polymerization techniques known in the art. For example, co-immobile buffer and enzyme polymer of the present invention are readily prepared as foams such a polyurethane foams. Other foams can be prepared from, for example, polyethylene (PEG)-modified enzyme and buffer. In such a polymer, enzyme and buffer can, for example, be bonded together to a functionalized PEG such as PEG diisocyanate. Representative co-immobile buffer and enzyme polymeric gels of the present invention include, but are not limited to, urethane, acrylate, and other types of gels wherein buffer and enzyme can, for example, be immobilized through covalent linkage.
The co-immobilized polymers of the present invention can be used in many different applications, including, but not limited to, reusable pesticide decontaminating and detoxifying pads for cleaning spills or equipment and polymer cartridges for detoxifying large volumes of contaminated pesticide water. In another application, buffer and hydrolase enzyme co-immobilized polymers of the present invention can be used to decontaminate and detoxify chemical nerve agents such as soman without loss of activity associated with pH. In that regard, degradation of soman results in high amounts of acid byproduct.
The present invention, along with the attributes and attendant advantages thereof, will best be appreciated and understood in view of the following detailed description taken in conjunction with the accompanying drawings.